Neuroprotective Effect of Carnosine Is Mediated by Insulin-Degrading Enzyme

l-Carnosine is an endogenous dipeptide that has high potential for therapeutic purposes, being an antioxidant with metal chelating, anti-aggregating, anti-inflammatory, and neuroprotective properties. Despite its potential therapeutic values, the biomolecular mechanisms involved in neuroprotection are not fully understood. Here, we demonstrate, at chemical and biochemical levels, that insulin-degrading enzyme plays a pivotal role in carnosine neuroprotection.


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suspended in DMSO at the final concentration of 5 mM and diluted in ice-cold cell culture medium DMEM/F12 (1:1) at the final concentration of 100 μM. Aβ1-42 samples were then incubated at 4 °C for 72 hours and immediately used to treat primary pure or mixed neuronal cultures or aliquoted and stored at -20 °C until their use.

Primary pure and mixed neuronal cultures
Cultures of pure cortical neurons were obtained from rats at embryonic day 15 as previously described. 3 Briefly, cortical cells obtained from cortices dissection were plated on 48-well plates precoated with 0.1 mg/ml Poly-D-lysine at a density of 2 × 10 5 /well. Neurons were grown in Neurobasal medium supplemented with B27, glutamine (2 mM), and the antibiotics penicillin (100 U/ml) and streptomycin (100 μg/ml). In order to avoid any proliferation of non-neuronal cells, Cytosine-Darabinofuranoside (10 μM) was added to each dish 18 hours after plating and was kept for 3 days before the next change of medium. As confirmed by using immunocytochemistry for glial fibrillary acidic protein and flow cytometry for neuron-specific microtubule-associated protein 2, this method allows to obtained neuronal cultures with a very high purity (>99%). 1,4 Primary mixed neuronal cultures (35-40% of neurons; 60-65% of astrocytes and microglia) were obtained from rats at embryonic day 15 as previously described. 1,3 Cells were plated at a density of 2 × 10 5 /well on 48-well plates and grown into MEM supplemented with HS (10%), FCS (10%), glutamine (2 mM), and glucose (6 mg/ml). In order to avoid any proliferation of non-neuronal cells, Cytosine-Darabinofuranoside (10 μM) was added to each dish 5 days after plating and was kept for 3 days before the next change of medium.

Measurement of cell viability and cell death by the MTT and trypan blue exclusion assays
Pure neuronal cultures were treated at 7 days in vitro with Aβ1-42 oligomers (1 μM) for 48 hours both in the presence and in the absence of carnosine (Car) (10 mM). Neuronal cell viability was measured by the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay. At the end of the treatment cells were incubated with MTT (0.9 mg/ml final concentration) for 2 hours at 37 °C. Following this incubation step, the MTT solution was removed and the formazan crystals were melted by using DMSO. As a last step, 200 µL coming from each type of sample were transferred to a 96-well plate and the absorbance at 569 nm was read using a plate reader (Spectra Max M5, Molecular Devices, Sunnyvale, CA, USA). Untreated neuronal cells were used as controls.
In the case of primary mixed neuronal cultures, cells were treated with Aβ1-42 oligomers (1 μM) for 48 hours both in the presence or in the absence of Car (10 mM). The possible neuroprotective activity against Aβ1-42-induced toxicity played by IDE was indirectly investigated by using the specific inhibitor of this enzyme, 6bK, at the final concentration of 250 nM. At the end of the treatment, the toxicity induced 4S by Aβ1-42 oligomers and the neuroprotection exerted by Car were quantitatively assessed by trypan blue exclusion assay. 1 Cell counts was performed in three to four random microscopic fields/well.

Statistical analysis
Graphpad Prism (Ver. 8, San Diego, CA, USA) software was used for statistical analysis. For multiple comparisons, one-way ANOVA followed by Bonferroni's post hoc test was employed. For all the experiments, the statistical significance was set up at p values lower than 0.05.

Study Approval
The study in neuronal cultures was authorized by the Institutional Animal Care and Use Committee  *** ***

Dynamic light scattering measurements
The measurements were performed with a Zetasizer Nano ZS (Malvern Instruments Ltd., UK) instrument equipped with a He-Ne laser. DLS measurements were run on disposable, temperature-resistant microcuvettes by using optimal measurement times and laser attenuation settings. All samples were

HPLC -Mass Spectroscopy
The enzymatic digestions of Human Ins and Aβ1-40 were performed according to the following methods: A solution of Ins (20μM) or Aβ1-40 was incubated with IDE (180nM) in PBS buffer solution (pH 7.3) at 37°C inside a controlled temperature incubator. The experiment was monitored over time, in the absence or in the presence of Car 100 μM and 1mM. Lastly, 10% TFA was added for each sample in order to quench the IDE activity. The TFA final concentration was the aliquots is 0.2%.
The kinetic degradation of Ins and Aβ1-40 and the analysis of their fragments have been obtained through the use of HPLC-Mass Spectrometry (See Figure 4S and Table 1S). The mobile phase consists in a mixture   respectively. It is important to highlight that Car did not affect the overall fluorescent response, as it is reported in Figure 6S.

Functionalization of gold sensor chip
The gold sensor chip was functionalized with the Lomant's reagent in DMSO (Lomant's reagent/DMSO stoichiometric ratio = 2) under a nitrogen atmosphere for five days. 6 After five days, the sensor was washed with fresh DMSO, ultrapure water, ethanol and lastly it was dried out with a N2 flow.

Immobilization of Insulin Degrading Enzyme (IDE)
The immobilization of IDE was carried out by fluxing a solution of 100nM of IDE in PBS (pH = 7.4) on a functionalized gold sensor chip in parallel configuration (only on the sample channel) at 15µl/min for

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The immobilization occurs via peptide bonds between the ammino groups of IDE and the carboxylic groups of Lomant's reagent with the release of N-hydroxysuccinimide group.
Once the immobilization of IDE was reached, a solution of Ethanolamine-HCl (1M, pH = 8) was flowed on the sensor twice in serial configuration (both sample and reference channels) at 15µl/min for 15 minutes in order to deactivate all the residual active sites on the surface.
In order to evaluate the number of IDE molecules immobilized on the surface, the SPR angle shift due to the enzyme immobilization has been measured and, in our experimental conditions, resulted to be 0. Moreover, the adlayer thickness (d) of IDE was calculated from the following equation: where θ corresponds to 9.104 x 10 11 molecules cm -2 and N is the bulk number density of the adsorbate. This latter is obtained by dividing the bulk density of adsorbate ρ (ρ = 1.30 g cm -3 ) 7 and the Avogadro's number multiplied by the molecular weight.
Thus, taking in account the previous formula, the IDE adlayer thickness (d) is 1.37 nm.
In order to minimize the aspecific interactions between insulin (Ins) molecules and the reference channel, a Bovine Serum Albumin (BSA) solution (100 µM) was flowed on the sensor in parallel configuration (only on the reference channel) at 30µl/min for 7 minutes of injection. Indeed, the common blocking agent used for activated ester groups is Ethanolamine, but a further treatment of the sensor with a BSA solution allows a more thorough deactivation of the reference channel surface. 8,9 Analogously, the same procedure was applied for the immobilization of IDE   Table 1 of the manuscript.